May 15, 2015 (Vol. 35, No. 10)
Andrew Birnie Ph.D. Business Development Specialist PCRmax
New Developments in PCR Instrumentation Help Researchers Meet Modern Regulations
Within any investigation or report submitted for publication, it is crucial to ensure that any process included is undertaken with complete accuracy and reliability. This is true of any scientific technique including but not limited to qPCR.
As demonstrated by the mistaken link between MMR vaccine and developmental disorders, which led to many children not undergoing vaccination, poor qPCR data can have huge implications. The implementation of the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines, therefore, set out the minimum information necessary for evaluating qPCR experiments.
However, meeting these guidelines presents its own challenges. Researchers must be able to demonstrate that multiple aspects of their investigation were undertaken correctly and present all the additional relevant data. As well as this, there is a need to show that the instrument being used is up to the necessary standard. All this must be achieved on top of the time and cost restrictions present in any modern laboratory.
Among the points listed in MIQE guidelines are PCR precision, limit of detection, and linear dynamic range. These features all ensure that the instrument is capable of generating data to the level of accuracy and reliability required for the results to be viable.
The latest technologies available in commercial instruments are capable of delivering the results required to meet these standards, while also delivering time and cost savings to laboratories. By implementing new materials and innovative designs, it is possible to produce instrumentation that makes meeting MIQE guidelines simple.
The below investigation demonstrates how these advancements meet modern requirements and presents the relevant data.
1) PCR Precision
Temperature or concentration differences, as well as stochastic variation, are just some of the explanations for variation in qPCR results. Usually, precision in qPCR differs with concentration and decreases with the copy number. However, researchers must be sure that any observations or variations recorded are results of the samples, and not the instrumentation. This is where new technologies are starting to play a major role.
For the highest accuracy, temperature must remain uniform across the entire heat block, ensuring that all samples are heated equally. This has been achieved recently through the implementation of a highly conductive hollow silver thermal block filled with a thermally conductive fluid. The fluid is rapidly circulated through the block by paired agitators powered by high-efficiency brushless motors, and the constant circulation evenly distributes the heat throughout the block.
Gage R&R (Gage Repeatability & Reproducibility) studies are valuable six sigma tools for evaluating and identifying variables in technician and instrument performance that may impact analyses. The study in Figure 1 was conducted using four randomly selected thermal cyclers of the same type (Eco system, PCRmax), with the same experiment performed in triplicate using four real-time fluorescent chemistries (SYBR, FAM, HEX, and ROX).
The SYBR assay was performed with 5,000 copies of human genomic DNA template in a 10 µL reaction volume, while the additional assays were performed with 5 ng and 10 ng of Human Reference cDNA templates in 10 µL reaction volumes. Each plate was run with the same PCR thermal profile as recommended by the master mix manufacturer.
The system demonstrated high precision within and between all four instruments. The mean Cq (cycle of quantification) value for the SYBR assays was 21, 27, with the standard deviations between the instruments reaching from 0.06 to 0.08. Such precision allows for a statistically significant call to be made between samples with as little as 20% different in target expression levels, which run in duplicate in any position across three plates. Similar precision can be seen in both the 5 ng and 10 ng samples using FAM-, HEX-, and ROX-based reporters.
With this level of precision, researchers can be sure of producing the correct results on the first run. This not only causes a marked increase in accuracy, but it reduces the requirement for numerous replicates and improves confidence in the data.
2) Limit of Detection
The Limit of Detection (LOD) is defined as the lowest concentration at which 95% of the positive samples are detected. In other words, within a group of replicates containing the target at concentrations at the LOD, no more than 5% failed reactions should occur.
The use of an ALC (adaptive LED control, shown in Figure 2) high-performance optical system has been shown to make a real difference in delivering precise and sensitive detection. When selecting a qPCR instrument, it is recommended to look for one with two panels of 48 fixed LEDs in order to provide excitation energy of distinct spectra, enabling broad range excitation. With these instruments, each of the LEDs will illuminate a specific well location, eliminating the optical distortion created by most stationary optical systems. HRM analysis protocols are supported by continuous data acquisition during the melt for increased data collection and reduced run times.
This new development means there is no uncertainty over whether unknowns have saturated, are bleaching through light to neighboring wells and maximize the wide dynamic range—users can enjoy complete confidence in their results.
3) Linear Dynamic Range
Finally, MIQE guidelines also state the dynamic range over which a reaction is linear (the highest to the lowest quantifiable copy number established by means of a calibration curve) should also be included in any submissions. The dynamic range needs to cover at least three orders of magnitude, depending on the template used for generating calibration curves, and ideally should extend to 5 or 6 log10 concentrations. The calibration curve’s linear interval must include the interval for the target nucleic acids being quantified.
Because lower limits of quantification are usually poorly defined, the variation at the lowest concentration claimed to be within the linear interval should be determined. Correlation coefficients (R2 values) must also be reported. In the below assay (Figure 3), for example, the R2 was 0.999—an extremely high result, which suggests that there is little variation and ambiguity within this investigation.
Since the implementation of the MIQE guidelines, it has been clear what is required of researchers in order to ensure investigations are valid. All data submitted for scientific review has to be demonstrated to be accurate and reproducible, and this can only be achieved when the instrumentation used is up to modern standards.
Manufacturers of qPCR instrumentation are increasingly developing technologies which make meeting these guidelines simple. In recent years, a number of innovations have been seen which meet these goals. The above demonstrations show how it is now possible for researchers undertaking qPCR to meet and surpass all required guidelines, with no compromise to efficiency, cost, or speed.